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Rev. sci. tech. Off. int. Epiz., 1991, 10 (3), 813-846 Survival of pathogenic micro-organisms and parasites in excreta, manure and sewage sludge D. STRAUCH * Summary: The causative agents of many infectious diseases are excreted by the faecal route and also with other excretions or secretions of the body. Some pathogens are also excreted from clinically healthy animals, from those with latent infections and in cases of transmissible multifactorial diseases. In all types of livestock housing, the pathogens finally reach the floor with the installations for collecting manure as a solid or liquid. Under these conditions livestock owners do not realise that manure may contain pathogens, and therefore do not take precautions against possible spread of diseases by utilisation of manure. The pathogens do not survive very long in stored farmyard manure because of the temperatures and biological and biochemical activities prevailing in the middens. But the conditions in slurry are different because the temperature does not rise and biochemical activity is low. Therefore the pathogens survive for rather long periods in slurry. To avoid disease transfer by utilisation of manure and slurry as fertilisers, certain precautions are necessary and these are described in detail. The agricultural utilisation of municipal sewage sludge is common in many countries. However, these sludges contain pathogens which are excreted by the human population served by the sewers and sewage treatment plants. In the sewage purification processes most of the pathogens are reduced in number but not completely eliminated. They are enriched by sedimentation processes in the sewage sludge. To protect the livestock of farms utilising sewage sludge as fertiliser or for amending soils it is necessary to sanitise hygienically dubious sludges prior to their use. The epidemiological aspects of agricultural sludge utilisation are discussed and details of the available sanitation technologies are given. KEYWORDS: Bacteria - Disinfection - Epidemiology - Livestock - Manure Parasites - Sewage sludge - Slurry - Tenacity - Viruses. INTRODUCTION A n i m a l excreta a n d m a n u r e In this chapter the term 'excreta' refers t o faeces a n d urine and the term ' m a n u r e ' , which will be used as a general t e r m , refers to animal excreta in any f o r m , with or without b e d d i n g and dilution water. ' F a r m y a r d m a n u r e ' (FYM) refers t o a mixture of excreta together with substantial quantities of bedding materials (e.g. straw, w o o d shavings, sawdust, peat) dense enough to be handled as a solid. 'Slurry' is a mixture of faeces and urine which can also contain cleaning water, rain water, small quantities of bedding material a n d spoiled feed particles. * Institute of Animal Medicine and Animal Hygiene, University of Hohenheim (460), P . O . Box 70 05 62, 7000 Stuttgart 70, Federal Republic of Germany. 814 Several hundred diseases are transmitted among animals and more than 150 of them can be passed on to h u m a n beings as zoonoses (S.L. Diesch, unpublished findings). The pathogens causing most of these diseases are either excreted directly by the affected animals or indirectly spread into the environment by vectors such as nasal, pharyngeal, vaginal, placental or lochial secretions, faeces, urine, milk, sperm, dermal and mucosal desquamations and secretions, carcasses, blood, slaughterhouse offal, meat and meat products, milk and milk products, eggs and egg products from dairies, solid and liquid manures (FYM, slurries). In all of these conditions the pathogens are excreted and end up during indoor keeping of livestock on the floor and thus in the manure, even if the pathogen is not excreted by the faecal route. The floor of animal houses, with their installations for collecting faeces and urine, thus acts as the collecting basin for all pathogens which are spread by the animals of a given establishment. In every case of a clinically diagnosable transmissible disease, dung and slurry must be considered as infectious and an inanimate vector for the pathogen. To make matters worse there is, especially in the case of salmonellosis, a tendency for these pathogens to occur in slurries of livestock which have never shown any sign of clinical illness. After the second and third microbiological examination of the same slurries, the salmonellas are not isolated again (33, 42, 47). Similar conditions may also be found in the course of subclinical infections with other pathogenic agents. But such incidents risk developing into multifactorially caused diseases, whereby normally harmless infections may lead to clinically recognisable symptoms of disease released by nonmicrobial factors. When they exist in a population for months or even years a gradual accumulation of infectious agents will result, if further factors are added, in the outbreak of a dangerous multifactorial disease. Today this type of disease holds a special threat for larger and specialised livestock establishments with a lack of population heterogeneity, such as premises for fattening of only pigs or calves or broilers. The danger lies in the fact that in most cases the owner of such an establishment does not recognise the infection and therefore does not take any action to prevent a spread of the pathogens in his surroundings. Hence he is not informed about the infective potential of the excretions of his animals and the manure to be utilised o n his fields. For more than two decades salmonellosis has spread steadily among man and animals in many countries. In some areas, more than one-third of pigs and more than one-half of broilers produced for slaughter are infected with salmonellas. This is the reason why manure from larger production units generally should be regarded as infected, and disposed of by means of certain safety measures. The spread of many classical h u m a n diseases such as cholera, typhoid fever or bacillary dysentery has been controlled by insulating man from human excreta through improvements in personal hygiene and the use of sewage and water treatment processes. In areas of the world where such improvements have not been made or where available systems are inefficient these diseases remain endemic. The treatment of animal excrements with methods used for the purification of municipal sewage is, with the exception of a few gigantic intensive units in various parts of the world, generally considered too expensive and unsuitable for farm animal excreta. It is therefore necessary to find simple methods to minimise the environmental hazards of ' n o r m a l ' manure which is only possibly infected, and more sophisticated methods to treat manures which are certainly infected in order to render them innocuous for further utilisation as fertilisers or for other purposes (58). 815 Sewage sludge The terms 'sewage sludge' and 'sludge' used in this chapter are defined as all aqueous matter which is separable from municipal wastewater, except screenings, sievings and grit. Sludge consists of the particulate matter in raw sewage (wastewater) like faeces, food leftovers, paper, cellulose (diapers, etc.), the excess sludge of biological treatment steps and sludge from trickling filters — which is to say, sludge consists mainly of organic material. The water content is between 90-99%. Sludge is a good growing medium for bacteria as it rather quickly putrefies and needs to be stabilised before further utilisation or deposition. It is known that sewage and sewage sludge from municipal wastewater treatment plants do contain pathogenic agents. These germs derive from humans who use the sewerage systems and who suffer from acute or latent infections or from known and often unknown permanent excretors of pathogens (e.g. salmonellosis). The spectrum and quantity of pathogens are extended by other sources connected to the sewerage system, like hospitals, abattoirs, livestock markets and related activities. It is known too that a certain part of the h u m a n population, companion animals and livestock are always afflicted with an infectious disease. In view of the fact that it is nowadays possible to isolate pathogens from raw municipal sewage without great effort, it seems pointless to speculate about the percentage of infected individuals. Since a variety of fairly intangible factors play a role, the estimates vary considerably around the factor 10 (0.5-5% of the population using the sewers). Pathogens are excreted from infected individuals via faeces, urine, secretions or excretions of the nose, pharynx, vagina, mucous membranes and skin, depending on the type of infection, and reach the sewage treatment plants from sewers and sanitary installations in homes. It is therefore understandable that for epidemiological surveys some authors consider microbiological examination of wastewater as reflecting the epidemiological situation of the population in certain catchment areas. Sewage sludges usually do contain considerable amounts of pathogens even after the purification processes (57). Farmers in industrialised countries are increasingly exercising restraint in the agricultural utilisation of sewage sludge, because they feel it a threat to their livestock, especially when the animals are grazing or fed with green feed. In view of the steady increase of latent infections with salmonellas and agents of multifactorial diseases in the intensive production of poultry, pigs and calves, farmers want to seal their farms off from further danger of infection. In recent years many farmers have come to believe that their soil was being slowly but surely turned into the rubbish bin of the nation. The following pages will discuss whether and to what extent fears regarding the danger for livestock of infection from sewage sludge are justified, and what measures can be taken to eliminate or minimise the possible dangers. P A T H O G E N I C A G E N T S IN E X C R E T A A N D MANURE Bacteria Theoretically, the bacteria involved in cases of bacterial infection can occur in the excreta and m a n u r e of infected animals. An expert group of the Commission 816 of the E u r o p e a n Communities (CEC) has listed those bacteria (1) which may be of particular concern for animal a n d / o r h u m a n health under European conditions when they are present in animal excreta and manure (Table I). TABLE Bacteria of epidemiological concern I in livestock excretions and manure * (1) Salmonella spp. Escherichia coli (including enteropathogenic strains and normal gut E. coli multiply resistant to antibiotics) Brucella spp. Bacillus anthracis Leptospira Erysipelothrix rhusiopathiae Mycobacterium spp. (in particular M. tuberculosis, M. bovis, M. avium complex, M. paratuberculosis and the atypical mycobacteria) Treponema Chlamydia Rickettsia spp. hyodysenteriae spp. spp. * Outbreaks involving some o f these organisms are subject to specific regulations in different countries which include control o f the movement and utilisation o f excreta and manure f r o m infected enterprises Since a b r o a d variety of pathogenic bacteria can be excreted by infected animals, it may be assumed that a similar number of organisms can at some time be found in their excretions and manure. Under practical conditions the number will be limited by many factors, and the variety of pathogens actually isolated is comparatively small. T h e kind of bacteria isolated will vary with the geographical location of the farm and the animal species. Also the physical and chemical composition of the manure may determine the types of pathogen present. Thus leptospires, which are sensitive to extremes of p H , may be isolated only from slurries with a p H a r o u n d neutral. Isolations and occurrence will also depend upon the age of the slurry and its dry matter content, as well as on the number of organisms gaining access to the m a n u r e , which will obviously affect the possibility of pathogens being present and the outcome of attempts at isolation. Another important factor is the ease with which small numbers of pathogens may be isolated. M a n u r e naturally contains an excess of 1010 bacteria per ml from which the pathogenic bacteria must be separated. Small numbers of Enterobacteriaceae, e.g. salmonellas, may be isolated because very sensitive enrichment and identification techniques are available. Other bacteria like those causing anthrax, brucellosis, mycobacteriosis and leptospirosis will be isolated only if larger numbers are present and when reliable isolation techniques for the respective pathogen are available. Therefore the number of occasions on which a pathogen has been isolated from m a n u r e obviously cannot give a true reflection of its occurrence, and it seems justifiable t o assume that any organism which is voided in the faeces and urine of animals or other body fluids and excretions may be found in manure. Very few surveys have been carried out to assess the actual prevalence of pathogens, and those which can be referred to have usually concentrated on salmonellas, although other organisms have occasionally been isolated (6, 2 1 , 35, 36, 37, 40). 817 The number of pathogens contained in cattle and pig m a n u r e is probably low. Surveys in the United Kingdom did not reveal concentrations of salmonellas above 10 per ml. But the concentrations may also be higher because even apparently healthy cattle may excrete up to 10 salmonellas per gram of faeces (34) and a similar n u m b e r of leptospires may be excreted in the urine of infected cattle (27). Excretion at this level by only a few animals of a herd could render m a n u r e a potent source of pathogenic organisms. Pathogens have also been found in poultry m a n u r e but such reports are few. This may relate to the manner in which poultry waste is collected. It usually tends to be a solid or semi-solid material which remains aerobic and is readily composted. Pathogens like salmonellas, colibacteria, pasteurellas and hemolytic streptococci are killed rapidly under these conditions (48). Others like listerias or clostridias m a y withstand the adverse conditions involved. The type of waste may also affect the longevity of organisms, since salmonellas are killed m o r e rapidly in built-up than in fresh litter, and this may affect the frequency of isolation (58). In a recent investigation the tenacity of Salmonella typhimurium in faeces of laying hens was studied in five different types of housing (intensive floor husbandry without litter, floor h u s b a n d r y with litter, battery without aeration of the droppings belt, battery cages with aeration of the droppings belt, and sloping floor) and two types of middens (covered midden for the battery cages without belt aeration, covered midden for the battery with belt aeration). The tenacity of 5. typhimurium varied between 2 and 175 days, depending on the h u s b a n d r y system (2 days in the sloping floor system and 175 days in the midden from the battery with aerated droppings belt). Relevant environmental factors were temperature and the dry matter content of the m a n u r e (50). 2 7 Viruses Information about the occurrence and tenacity of viruses in animal excrements is not as a b u n d a n t as in the case of bacterial infections. According to a survey on the excretion of viruses by farm livestock, based on research in N o r t h America (Table II), it seems a likely supposition that these findings are valid not only for N o r t h America but, with local variations, for most parts of the world. A n o t h e r compilation gives details about the presence of various animal viruses in faecal matter (Table III). But as these two tables show, a considerable number of virus genera are already k n o w n to be excreted in the faeces of livestock and other animals. Only in recent years have improvements in the isolation techniques for viruses from heavily contaminated material led to remarkable successes in identifying viruses in faecal matter. This development has m a d e it possible t o consider excrements and the spent air of animal houses in epidemiological discussions. Enteroviruses are relatively tenacious in slurry. Quantitative investigations in slurry samples with direct isolation of viruses showed a virus titre of about 10 T C I D per litre, whereas in samples which yielded enterovirus only after concentration, about 50-100 T C I D 5 0 per litre were found (19). Infections of pigs with parvovirus are not infrequent and the survival of this virus in slurry and sewage is documented. Transmissible gastro-enteritis virus is relatively sensitive to environmental influences, just as are other coronaviruses. As for the influenza viruses of pigs, these have low tenacity. Their excretion by the faecal route is considered to be unlikely, but nevertheless they can reach the floor and thus the m a n u r e of stables with the nasal excretions of infected pigs. Besides typical pig strains, infections of pigs with 6 5 0 818 TABLE Viruses excreted by farm II livestock in North America (18) Host species Non-enveloped viruses Enveloped viruses Cattle Bovine enterovirus Bovine adenovirus Reovirus Reovirus-like Bovine parvovirus Bovine rhinovirus Bovine papillomavirus Infectious bovine rhinotracheitis Malignant catarrhal fever Bovine mammillitis Bovine virus diarrhoea Bovine Coronavirus Parainfluenza type 3 Respiratory syncytial virus Rabies Vesicular stomatitis Cowpox Paravaccinia Sheep Ovine enterovirus Ovine adenoviruses Rcoviruses Porcine parvoviruses Contagious ecthyma Maedi-visna Malignant catarrhal fever Transmissible gastro-enteritis Hemagglutinating encephalitis Hog cholera Swinepox Poultry Avian enteroviruses Avian adenoviruses Avian reoviruscs Newcastle disease Avian influenza Infectious laryngotraeheitis Infectious bronchitis Avian leukosis Marek's disease Fowlpox Viral arthritis h u m a n strains o f i n f l u e n z a virus are m o r e frequently o b s e r v e d in c o n n e c t i o n with e p i d e m i c s in the h u m a n p o p u l a t i o n . A n t i b o d i e s w h i c h react with the h e m a g g l u t i n i n s o f s w i n e i n f l u e n z a virus are present in m a n y h u m a n sera. T h e h u m a n strains o f this virus can persist for years in the pig p o p u l a t i o n . Pigs m a y thus be considered a possible reservoir for h u m a n strains o f i n f l u e n z a virus. F r o m k n o w l e d g e o f the o c c u r r e n c e and survival o f viruses o f h u m a n origin in w a s t e w a t e r , s l u d g e , soil a n d o n plants w h i c h were fertilised with s e w a g e ( m o r e than 100 different strains o f enteroviruses were isolated), it can be a s s u m e d that the agents o f m a n y viral diseases o f d o m e s t i c a n i m a l s are excreted in the faeces and urine of infected a n i m a l s , a n d therefore m u s t o c c u r frequently in m a n u r e (58). Parasites T h e prevalence o f p a t h o g e n i c p r o t o z o a , h e l m i n t h s a n d a r t h r o p o d s in excretions a n d m a n u r e has been d i s c u s s e d under E u r o p e a n c o n d i t i o n s (13). Cattle faeces are regularly c o n t a m i n a t e d with large n u m b e r s o f Eimeria o o c y s t s and trichostrongyle 819 TABLE III Presence of various animal viruses in faeces, (52, modified) Viruses excreted primarily in faeces Enteroviruses Reoviruses Rotaviruses Bovine virus diarrhoea Transmissible gastro-enteritis of pigs Calf diarrhoea Coronavirus Rinderpest Parvoviruses Adenoviruses Viruses clearly present in faeces Aujeszky's disease Foot and mouth disease Swine vesicular disease Coxsackie Classical swine fever African swine fever Viruses probably present in faeces Vesicular exanthema Rift Valley fever Viruses unlikely to be present in faeces Bluetongue African horse sickness Louping ill Maedi-visna Hemagglutinating encephalomyelitis Vesicular stomatitis Rabies Swine influenza Parainfluenza 3 Herpes group: infectious bovine rhinotracheitis Contagious pustular dermatitis Poxviruses secretions and excretions Virus also present in high titre in: Respiratory secretions. Respiratory secretions Maximum amount of virus found in: Respiratory secretions, saliva Vesicular epithelium Vesicular epithelium Blood Blood Maximum amount of virus found in: Vesicular epithelium Blood Maximum amount of virus found in: Blood Blood Blood Respiratory secretions Respiratory secretions Vesicular epithelium Saliva Respiratory secretions Respiratory secretions Respiratory secretions Pock epithelium Pock epithelium eggs. Quantitatively these constitute the prime c o n t a m i n a n t s in cattle effluents. Fasciola eggs, though less frequently present, are also a considerable risk as they are very resistant a n d may multiply if they find a suitable habitat where a snail host may become infected. Other contaminants create minor hazards due to their low resistance (eggs and larvae of Strongyloides, Dictyocaulus larvae), the need to enter into an intermediate host (Dicrocoelium, Moniezia) or just their relatively low prevalence (Toxocara vitulorum) (Table IV). 820 TABLE Prevalence and resistance of parasitic agents in cattle and their potential hygienic hazard (13) spp. Helminths Trichostrongylid spp. Strongyloides papillosus Oesophagostomum spp. Fascioia hepática Dictyocaulus viviparus Trichuris spp. Dicrocoelium dendriticum Moniezia spp. Toxocara vitulorum Resistance ( ) Priority as a hygienic hazard + + + + + + 1 + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 2 + + + ? 1 Arthropods Psoroptes Chorioptes Sarcoptes a) + + + regular h) + + + high + + frequent + + intermediate effluents Prevalence ( a) Parasite Protozoa Eimeria IV b 3 + occasional + low It was found in Ireland that 94-96% of slatted-floor slurry samples contained trichostrongylid nematodes, mainly Ostertagia ostertagi and Cooperia oncophora, in contrast to FYM samples, only 7 9 % of which had these nematodes (44). Another comparison showed similar conditions in Sweden, where 4 1 , 0, 6, 8 and 2 % of F Y M samples were carriers of trichostrongylids, Eimeria s p p . , Moniezia benedeni, Trichuris s p p . and Nematodirus helvetianus respectively, c o m p a r e d with contamination rates of slurry samples of 67, 6, 17, 8 and 19% (46). Pig faeces regularly contain Eimeria oocysts and frequently eggs of several gastro intestinal nematodes. A m o n g these, Ascaris eggs and coccidial oocysts are the most dangerous from the qualitative point of view due to their high resistance. Eggs of other species are less important though they may occasionally give rise to infections (Table V). Broiler and hen litter will normally be contaminated by oocysts of various Eimeria species. As these are highly resistant, they rank first among the potentially diseaseinducing contaminants. Although less frequently present in poultry litter, eggs of Ascaridia and Heterakis are dangerous on account of their high resistance. Other contaminants may keep the life cycle of a parasite going; however, they will give rise to serious trouble only occasionally (Table VI). The priority of a contaminant according to its prevalence in the effluent, and its innate properties of resistance and survival potential, may be modified not only by 821 TABLE Prevalence and resistance of parasitic agents in pig and their potential hygienic hazard (13) Parasite Protozoa Eimeria Balantidium Helminths Ascaris Oesophagostom Strongyloides Hyoslrongylus Trichuris Fasciola V effluents Prevalence (a) Resistance (b) Priority as a hygienic hazard + + + + + + + + 2 + + + + + + + + + + (+) + + + 1 3 + + + + + + + + + urn Arthropods Sarcoptes Haematopinus + + a) + + + regular + + frequent + occasional b) + + + high + + intermediate + low TABLE 5 4 ? VI Prevalence and resistance of parasitic and hen effluents and their potential (13) agents in hygienic broiler hazard Prevalence ( Resistance ( Priority as a hygienic hazard Protozoa Eimeria Histomonas + + + + + + + 1 Helminths Asear id ia Heterakis Capillaria + + + + + + + + + + + + + + 2 2 Parasite a) Arthropods Dermanyssus a) + + + regular + + frequent b) + + + high + + intermediate c) in Heterakis eggs + b) ? + occasional + low the conditions in which it is kept or stored after having been shed by the h o s t , but also by its subsequent use. T h e hygienic h a z a r d , for instance, of pig slurry is very 822 different depending on whether it is used as fertiliser on arable land, as fertiliser on sow or cattle pastures, or as recycled feed for cattle. The variety of parasites will also be influenced by composition and age. The parasites discussed above and the information shown in the tables must be modified according to the geographical area where a problem arises. Fungi Animal wastes are not usually considered as a source of pathogenic fungi although solid manure may provide a good growth substrate for many species, and Petriellidium boydii was the dominant member of the mycoflora of manure samples isolated from three beef cattle feedlots in the USA. This organism causes mycotic abortion in animals, pulmonary allescheriasis in man and mycetomas in both man and animals. Two other fungal species capable of producing mammalian mycotoxins were also isolated (8). T E N A C I T Y O F P A T H O G E N S IN E X C R E T A A N D M A N U R E Bacteria It has been reported that salmonellas may multiply during storage of slurry, but it is generally accepted that most pathogens are reduced by storage. The reason is that pathogens are adapted to growing in the tissues of their host, and the environment of slurry with its comparatively low temperature and the presence of other antagonistic organisms is obviously not suited to their continued existence. The type of slurry, storage temperature and serotype of salmonellas may all affect the survival time, which also depends to a considerable extent on the number of salmonellas contained in the slurry at the beginning of storage. The greater the number of pathogens present at the beginning, the longer will be their time to extinction. Survival is also enhanced by reduction of temperature and an increase in solids content. Survival is greatest at temperatures below 10°C and in slurries containing more than 5 % solids (35). The influence of p H on the survival of salmonellas in various types of slurry has been studied. These and other observations show that differences in the viability of salmonellas exist, depending on the animal species from which the slurry is produced. The following relation can be postulated: survival is longest in cattle slurry, median in pig slurry, and shortest in cage manure from poultry and calf slurry (Table VII). There are many reports on the survival time of pathogenic bacteria in excretions and manure which cannot be discussed here but which deserve attention (2, 5, 6, 17, 20, 28, 40, 43, 45, 60). Viruses Information is scanty on the survival times of viruses in farm effluents. Some viruses survive in faeces of various kinds, depending on summer or winter temperatures: Aujeszky's virus 3-15 weeks, Borna virus 22 days, Marek virus 7 days, Teschen virus 3-25 days, African swine fever virus 60-160 days, foot and mouth disease virus 21-103 days (45). In very recent investigations which were made together with other experiments (50) it was found that Newcastle disease virus was reduced in winter faster in battery cage faeces (22 days) than in two floor pen systems and in the middens 823 TABLE Survival under VII of different salmonella types in animal natural conditions in storage facilities of (58) effluents farms Survival (days) 5. dublin S. typhimurium S. paratyphi B S. anatum S. Manchester S. gallinurumpullonun pH Cattle slurry Cattle urine Calf slurry Pig slurry Poultry slurry 49 177 157 210 180 65 58 57 73 84 12 29 22 26 33 39 39 39 47 47 28 8 57 44 7.5-8.0 7.8-8.0 - - - 7.0-7.7 8.4-8.8 9.0-9.4 14 for s t o r a g e o f the d r o p p i n g s from the battery s y s t e m s . In s u m m e r the results were totally different: 22 d a y s in the battery s y s t e m s but o n l y 11 days in the m i d d e n s o f the floor pen s y s t e m s ( 2 6 ) . M a n y m o r e i n v e s t i g a t i o n s h a v e c o n c e n t r a t e d o n survival o f viruses in h u m a n s e w a g e or soil and h e r b a g e treated with s e w a g e and s e w a g e sludge (6). T h e c a u s a t i v e a g e n t s o f m a n y viral diseases are excreted in t h e faeces and urine. A n u m b e r o f viral d i s e a s e s , particularly viral enteritis, s w i n e vesicular disease and f o o t a n d m o u t h d i s e a s e c o u l d be spread by slurry, a l t h o u g h such cases h a v e yet to be reported (35). T h i s m a y be d u e to the fact that s o m e o f these diseases are n o t i f i a b l e and t h e r e f o r e are subject t o specific and very stringent regulations w h i c h e m p h a s i s e the control o f m o v e m e n t and utilisation o f animal excretions from infected f a r m s , a n d usually are b a s e d o n biological ( ' d u n g p a c k i n g ' ) or c h e m i c a l d i s i n f e c t i o n o f solid a n d liquid m a n u r e s . Parasites M a n y parasitic i n f e s t a t i o n s o f farm a n i m a l s are transmitted by ingestion o f infective stages in materials, including pasture, contaminated with faeces. T h e viability o f parasite eggs a n d larvae varies e n o r m o u s l y . In T a b l e s VIII a n d I X the tenacity o f eggs o f Ascaris suum a n d eggs a n d larvae o f s t r o n g y l e n e m a t o d e s is listed. T h e results indicate that especially Ascaris eggs and certain larval stages o f trichostrongylids h a v e a rather l o n g survival t i m e a n d m a y give rise t o further i n f e s t a t i o n s o f a n i m a l s after m a n u r e has been s p r e a d o n crops or pasture l a n d . Helminth larvae are usually killed by c o m p o s t i n g o f faeces, but often remain viable in slurry during storage. T h e y are resistant t o d i s i n f e c t i o n , and c o n c e n t r a t i o n s o f lime in excess o f 5 % are required for effective d e c o n t a m i n a t i o n . Soil will still c o n t a i n infective larvae o n e year after s p r e a d i n g o f c o n t a m i n a t e d slurry. E v e n p l o u g h i n g is n o g u a r a n t e e o f d e s t r u c t i o n o f parasite eggs a n d larvae, a l t h o u g h in practice it is a g o o d w a y o f r e d u c i n g their n u m b e r s . S t r o n g y l e and t r i c h o s t r o n g y l e eggs p l o u g h e d i n t o soil c a n h a t c h a n d m i g r a t e t o the s u r f a c e , a n d infective t r i c h o s t r o n g y l e larvae can survive in soil for m o r e than a year. Parasite eggs and larvae are destroyed during ensilage as a result o f the high temperature and fall in p H , but they m a y remain viable 824 TABLE VIII Survival of eggs of Ascaris suum under different environmental conditions (12) Medium Conditions Dungstead manure Liquid manure Slurry Slurry Liquid manure Pig manure Sewage sludge Sewage sludge and pig manure Cattle slurry Cattle manure At least 5 0 ° C / 4 0 ° C In laboratory 8 ° C / 1 8 ° C Storage pit, summer Storage pit 4 ° C / 1 0 - 1 5 ° C / 1 8 - 2 6 ° C Storage pit Non-aerated storage pit Dried In storage pits Liquid manure Pig and cattle manure Pig slurry End of viability (days) Aerobic treatment 35-40° C Storage pit 22-27°C/anaerobic digester 55°C Licom-system 53°C Anaerobic digestion 52-54°C Aerobic-thermophilic treatment 55°C TABLE Survival 19/63 85/65 75 28 365 365 1,825 810 14 57/1 2 1 1 IX of eggs and larvae (L) of (12) Species Medium Conditions Trichostrongylus colubriformis Cattle slurry 18°C/8°C trichostrongylids End of viability (days) Egg: 2 6 / 6 4 L, + L : 4 / 4 L : 37/76 2 3 Cooperia punctata 18°C/8°C Egg: 2 6 / 6 4 L, + L,: 4 / 4 L : 37/76 3 Ostertagia ostertagi Cattle slurry + Cooperia oncophora Trichostrongylids Licom-system, Licom-system, Licom-system, Licom-system, 20°C 20°C 3°C 3°C summer 56°C summer 56°C winter 50°C winter 50°C Cattle slurry under slatted floor Cattle slurry through the whole year Cattle slurry through the whole year * Possibilities for further development apparently not tested Egg: 2 L: 4 Egg: 6 L: 6 Egg: 28 L: 28 Egg: 172 L: 160 3 3 3 3 90 92 28* 825 in hay for over a year. Fortunately parasitic infections are seldom fatal, except in young animals, and the likely effect of spreading infected slurry is simply an increased parasite burden on animals which are already infected (35). EPIDEMIOLOGICAL CONSIDERATIONS Conventional livestock units which employ bedding do not cause a special epidemiological problem because, if proper management procedures are carried out and enough straw is used, dungsteads develop temperatures high enough to destroy pathogens that may be present. This can be seen from the results of a Swedish researcher who found that in cattle herds with slurry systems infected with salmonellas, 3 5 % of slurry samples were positive for salmonellas, whereas in similar herds with straw bedding and dungsteads, only 6 % of samples from solid manure were positive. Five of these six positive samples, representing 6% of the study, were from the surface of a deep litter bed, which can be regarded as a badly managed midden. F r o m this point of view the result is even more favourable for solid m a n u r e (62). The relative safety of this procedure is demonstrated by its being stipulated in many countries in the official provisions of dung disinfection for the control and eradication of notifiable infectious diseases, by means of what is called 'dung packing'. After three weeks the dung is considered to be disinfected and can be used without restriction. Also in Sweden, it was found that in FYM dungsteads the temperatures were usually below 30°C. The reason is that nowadays the 'average' Swedish cow excretes daily 20 kg of faeces and 5 kg of urine more than 25 years ago, and that the a m o u n t of straw for bedding used per cow and day has decreased from 4-6 kg to 0.5-1.5 kg. Therefore the middens have too much moisture content and the composting process becomes anaerobic, which results in a considerable reduction of the temperature, so that the disinfection of such dung is no longer ensured (49). Since similar conditions were found in German farms, a new technique for composting FYM was developed which combines composting with use of granulated quicklime. By this method a reliable disinfection of FYM can be achieved (9, 5 1 , 59). As stored slurry is usually anaerobic, no spontaneous generation of heat that could entail the destruction of pathogens will occur in that medium either in summer or winter. Therefore slurries containing pathogens always pose a health hazard to the animals of the particular farm and its neighbourhood. A certain degree of "selfdisinfection" during storage of slurry and the adverse influences of the environment after spreading on pastures and arable land underlie the fact that there are few reports about severe outbreaks of infectious diseases after utilisation of slurry, especially in grazing animals (54). As mentioned above, there is a slight possibility that the slurry of farms with clinically healthy animals may contain certain pathogens (e.g. salmonellas), which occur once or twice and then vanish again. To minimise this low risk the abovementioned expert group of the C E C elaborated Interim minimum guidelines in 1978 for the utilisation of " n o r m a l " slurries: 1. Slurry should be utilised on tillage crops (excluding crops consumption), wherever possible. for fresh 826 2. If slurry is spread on grassland, then use on pasture for conservation, wherever possible. If slurry is to be spread on grazing land, then: a) store all slurry for a minimum of 60 days before spreading b) allow 30 days before grazing c) graze with adult or non-susceptible animals. 3. Utilisation of slurry should be related to plant nutrient requirements (1). Several years later the same expert group discussed these guidelines again and approved them. It was found that they were of general application but should be modified according to circumstances, e.g. notifiable diseases where the procedures are laid down by law, particularly resistant pathogens like some parasites, transport of slurry to " s l u r r y / m a n u r e b a n k s " , and seasonal effects on survival of pathogens. Some recommendations have thus been added to the minimum guidelines: 4. Whereas storing slurry for 60 days in summer may be adequate, slurry produced in winter should be stored for at least 90 days. Storage for these periods requires two storage tanks. Prior to its removal, slurry inside animal houses has, of course, all the potential dangers of untreated slurry for stock in contact with it. Slurry from copper-supplemented pigs may be harmful to sheep grazing treated pastures. 5. Despite the wide variety of infective agents - bacteria, viruses, fungi and parasites - that can be present in slurry, there are few published records of disease transmission to animals or man through slurry, treated or untreated. The risk does exist, however, and it can be reduced to acceptable proportions if care is taken to treat and use slurry according to the minimum guidelines above. 6. Evidence was presented at the workshop to suggest that the guidelines are not sufficiently well known to farmers. Each country should ensure that steps are taken to make their contents widely known through the media - print, radio and television - as well as at agricultural meetings, shows and markets. 7. Despite the relative lack of unequivocal evidence that slurry transmits disease to man or livestock, it is recommended that the existing guidelines should be adapted to meet the particular needs of individual countries. Further research is needed: a) to determine the levels of contamination of pasture with pathogenic organisms that may result from treating it with slurry; and b) to evaluate methods for the disinfection of slurry without decreasing its value as a fertilising agent. 8. The workshop agreed that future activities should concentrate on communicable diseases rather than manure-associated problems. In accordance with the C E C , special emphasis should be placed on campylobacteriosis as an emerging disease and also on listeriosis. Attention should also be given to other zoonoses (2). Since the risks of infection associated with the spreading of slurry on farmland have not yet been clearly established, the safest procedure will be to aim at the best possible decontamination of infected slurry during the storage phase, e.g. before spreading. To achieve this purpose the decimal reduction time ( T ) for given pathogens under specific storage conditions has been used to calculate the holding time in batch storage of slurries (21). 90 827 S u c h use o f T90-values will require: 1. K n o w l e d g e o f t h e initial c o n c e n t r a t i o n in t h e slurry o f the p a t h o g e n i c a g e n t s concerned. 2. A d e c i s i o n as t o w h a t s h o u l d be regarded as the " a c c e p t a b l e final l e v e l " o f the p a t h o g e n s in the slurry after b a t c h s t o r a g e . 3. T h e presence o f m o r e than o n e storage facility o n a farm as a s a f e g u a r d against the i n t r o d u c t i o n o f freshly i n f e c t e d slurry d u r i n g batch s t o r a g e . A l t h o u g h o u r k n o w l e d g e is s c a n t y regarding the initial c o n c e n t r a t i o n s o f p o t e n t i a l l y p a t h o g e n i c bacteria in fresh slurry, several reports are available o n the c o n c e n t r a t i o n o f certain bacteria. S o far, n o d e c i s i o n has a p p a r e n t l y b e e n reached with respect t o an " a c c e p t a b l e final l e v e l " o f v a r i o u s p a t h o g e n i c bacteria in relation to d e c o n t a m i n a t i o n o f slurry. R e g a r d i n g Salmonella bacteria, the view m i g h t be t a k e n for practical p u r p o s e s that a reduction in n u m b e r s t o a level w h e r e , using special f a v o u r a b l e p r o p a g a t i o n m e t h o d s in the l a b o r a t o r y , for e x a m p l e , not o n e Salmonella c o u l d be d e m o n s t r a t e d per 10 ml o f slurry (i.e. log10 to the bacterial c o n c e n t r a t i o n : < - l ) w o u l d be a c c e p t a b l e . In t h e o r y , o f c o u r s e , it c a n n o t be e x c l u d e d that, under special circumstances, even minute a m o u n t s o f pathogens m a y give rise to cross c o n t a m i n a t i o n a n d / o r a risk o f i n f e c t i o n in a n i m a l s and m a n . D e s p i t e the o b v i o u s l i m i t a t i o n s in the applicability o f these results, their clear t e n d e n c i e s and relative u n i f o r m i t y within the individual bacterial species and e x p e r i m e n t a l g r o u p s give reason to a s s u m e that the T90-values f o u n d m a y be used p r o v i s i o n a l l y as a g u i d e l i n e for r e c o m m e n d e d h o l d i n g times for batch s t o r a g e o f infected slurry (21). A n e x a m p l e for the c a l c u l a t i o n o f required r e d u c t i o n levels is g i v e n in T a b l e X . TABLE X Storage time for slurry infected with S. typhimurium, calculated on the basis of T90- values for anaerobic and aerobic storage in winter and in summer (21) Storage time in weeks Winter T90 Summer T anaerobic aerobic anaerobic aerobic x 5.9 x 1.6 X 2.0 x 0.6 Required reduction 9 0 4 From 1 0 / m l to < 1/10 ml 30 8 10 3 From lOVml to < 1/10 ml 12 3-4 4 1-2 Similarly, Tyo-values for other p a t h o g e n i c agents m a y p r o v i d e an a p p r o x i m a t e i n d i c a t i o n o f r e c o m m e n d e d b a t c h s t o r a g e t i m e s for infected slurry in v a r i o u s situations. 828 These calculations may provide a basis for further consideration of needs and possibilities in setting up general or special guidelines for decontamination of infected slurry by batch storage. Reflections on ecological and technical-economic aspects of this and other possible decontamination methods should also be included in such considerations. Further investigations with this method are also necessary to prove its applicability to slurries infected with viruses and, perhaps, parasites. In the case of parasitic diseases, the epidemiological situation may be somewhat different from that of bacterial and viral diseases. Tables IV, V and VI provide lists of parasites which may occur in animal effluents in Europe. They require modification for parasitological situations in other parts of the world. In contrast to the epidemiological facts of infectious diseases caused by bacteria and viruses, parasites may need more than one host for their final development and they may have several larval stages, each differing in sensitivity to environmental influences. Furthermore, certain measures make it possible to interrupt chains of infection for parasites without disinfection of the inanimate vector, e.g. utilisation of pig slurry on cattle pastures and cattle slurry on pig pastures. A detailed account of the complex epidemiology of parasitic diseases is impossible in the context of the agricultural utilisation of manure and slurry. However, the recommendations in Table XI are based on the viability of some important parasitic eggs and larvae under certain environmental conditions listed in Tables VIII and IX (12). TABLE Hygienically XI safe utilisation of manure and slurry in from a parasitological point of view agriculture (12) Medium Stockpiled manure Fresh cattle slurry Fresh pig slurry Stored cattle slurry Stored pig slurry Poultry manure - Cultivated crops Vegetables Potatoes Sugar Corn Grain Green Meadow Pasture beets fodder + -- + + + + + + + + +! +! +! +! + + +! +! + + + + + + + + + + + + — - — +! +! + — — — +! +! + i i + Not applicable ! I f possible insert one cut for silage or hay winning + ! After topdressing with slurry do not feed fresh (dry or silage beforehand); avoid direct harvesting from the soil + Applicable without restrictions In some countries with very high densities of livestock in a given area, it is becoming common to store slurry jointly in large tanks, each with a capacity of several thousand cubic metres. This development is subsidised by the governments. These joint storage facilities raise the risks of disease spread, because the pathogen content of the 829 individual slurry is not k n o w n and the slurry mixture is distributed to all members of the facility. Even under very strict conditions it is not possible to eliminate this hygiene risk completely. Farms involved in such a joint storage system must therefore be willing to bear an acceptable risk. For this new development, important recommendations have been issued recently (32). In cases of notifiable diseases, all countries have special legal regulations on how the excreta and manure should be disinfected and handled thereafter. Solid manures are usually incinerated or composted, and slurries chemically disinfected. Since many of the chemicals which are used for slurry disinfection can be toxic for soil, plants or water, some ecologically desirable chemicals are listed in Table X I I . TABLE Suitable XII chemicals for the disinfection Disinfectant Dosage * (kg/m ) Reaction time (days) 9-15 25-40 8-12 40 4 4 4 4 3 Formalin 40% calcium hydroxide suspension Sodium hydroxide (NaOH) Peracetic acid 15% ** of slurry * Lower value: effective against enveloped viruses Upper value: effective against bacteria ** Only to be recommended for disinfection o f small amounts, due to the heavy formation o f foam Some larger livestock enterprises use anaerobic digestion of slurry to produce biogas. Most of these biogas plants are operated at mesophilic temperatures in the range of 30-35°C and the operators believe that pathogens are destroyed by the anaerobic digestion process. This opinion must be corrected because the mesophilic temperatures are not sufficient to destroy the pathogens. Only in the thermophilic range of 55°C can it be expected that the effluent of the digesters is disinfected. Therefore the effluent of biogas plants with mesophilic digestion has the same hygienic status as any slurry stored in a tank (30, 53, 66). Further ways of disinfecting m a n u r e by biological, biological-technical and chemical methods have been proposed, along with discussions on how to protect the environment from chemical disinfectants (3, 7, 40, 58, 60). P A T H O G E N I C AGENTS IN SEWAGE SLUDGE Bacteria As mentioned in the introduction, most pathogens enter sewage treatment plants from sewers. Their range depends largely on the epidemiological conditions in the particular region. 830 Table XIII lists the bacterial pathogens which may be encountered in the central part of Europe and which have been isolated from wastewater already. The table contains all current bacteria of greater or lesser importance for the epidemiology of infectious bacterial diseases. Although a given germ may be missing from this list, its occurrence in sewage can still not be excluded. It may be a constant inhabitant of the wastewater in certain regions (11, 14, 22, 31). TABLE Bacterial pathogens XIII to be expected in sewage (14, 39, 64) and sewage sludge Primary pathogenic Secondary pathogenic Salmonella s p p . Shigella s p p . Escherichia coli Pseudomonas aeruginosa Yersinia enterocolitica Clostridium perfringens Clostridium botulinum Bacillus anthracis Listeria monocytogenes Vibrio cholerae Mycobacterium s p p . Leptospira s p p . Campylobacter s p p . Staphylococcus Streptococcus Escherichia Klebsiella Enterobacter Serratia Citrobacter Proteus Providencia Viruses In the case of viruses t o o , a compilation of their regional range must vary. It is known that more than 100 viruses are excreted from humans in the faeces. Nearly every day new ones are added, some of which still await identification. They pass through the digestive tract in a typical manner and are excreted by infected persons in large a m o u n t s . The clinical signs which they provoke may have trivial to severe or even fatal consequences (22, 23, 24, 4 1 , 63, 64). A combination of increased awareness regarding the environment, improvements in laboratory techniques and the discovery of new viruses which are pathogenic for humans has led to an intensification of scientific activities, reflected in several hundred scientific publications on excreted viruses. There can be no doubt that a compilation of viruses excreted by humans (Table XIV) will undergo many changes in the coming years as a result of the characterisation of new viral agents and of a further differentiation of taxonomy. All of these viruses can, with regional differences, appear in the sewage. Viruses are also excreted with the faeces of animals, and viruses occurring in birds, dogs and cats can reach the sewers. Equally, it cannot be excluded that viruses derived 831 TABLE Viruses Virus group excreted by humans which in sewage and sewage (23, 41 modified) Number of types Enteroviruses Poliovirus Coxsackievirus A 3 24 Coxsackievirus B 6 Echovirus New enteroviruses Adenovirus Reovirus XIV 34 4 30 3 Hepatitis A virus can be sludge expected Diseases or symptoms caused Poliomyelitis, meningitis, fever Herpangina, respiratory disease, meningitis, fever Myocarditis, congenital heart anomalies, meningitis, respiratory disease, pleurodynia, rash, fever Meningitis, respiratory disease, rash, diarrhoea, fever Meningitis, encephalitis, respiratory disease, acute haemorrhagic conjunctivitis, fever Respiratory disease, eye infections Not clearly established Infectious hepatitis Rotavirus ? Astrovirus ? Calicivirus 7 Coronavirus Vomiting and diarrhoea 7 Vomiting and diarrhoea Common cold Norwalk agent and other small round viruses 7 Vomiting and diarrhoea Adeno-associated virus 4 Not clearly established but associated with respiratory disease in children from livestock may enter wastewater in various other ways. It is known that h u m a n viruses occur in domestic animals (Table XV). Unfortunately it remains an open question whether and to what extent the agricultural utilisation of municipal sewage and sludge contributes to this transmission. Parasites Parasitic infestations of humans and animals play a certain role. Under the conditions of Central Europe the number of parasites is rather limited compared with subtropical and tropical areas, where parasitic diseases may have top priority in frequency and importance. The range of parasites in Central Europe has certainly been enlarged by the influx of immigrant workers, refugees and resettled persons from various regions of the globe. But there are no indications that parasitoses have been transmitted to the native population to any considerable extent. Table XVI does not contain 'exotic' parasites but is restricted to those which belong to the usual range in Central Europe (29, 64). 832 TABLE X V Human viruses associated with (41) domestic animals Recovered from Virus identity Cattle Enteric viruses Poliovirus 1 Coxsackievirus A5 A6 A9 A10 A20 B3 B5 Echovirus 2 6 8 10 19 Reovirus 1, 2, 3 Respiratory viruses Influenza virus A 2 Mumps virus Cats Dogs Goat Horse + + + + Swine Infection produced * Cattle + + + + + + + + + + + Swine Dogs Dogs Dogs + + + + + Cats + + Cattle, dogs, cats + + + + Cats, swine Dogs * Infection determined by clinical or serological evidence Due t o biological peculiarities in the life cycle of most parasites, h u m a n s and animals play a similarly i m p o r t a n t role as intermediate and final h o s t s . The developing and final stages of these parasites reach the sewage directly from faeces. TABLE X V I Parasites to be expected in sewage (29, 64) and sewage sludge Protozoa Cestodes Nematodes Entamoeba histolytica Giardia lamblia Toxoplasma gondii Sarcocystis spp. Taenia saginata Taenia solium Diphyllobothrium latum Echinococcus granulosus Ascaris lumbricoides Ancylostoma duodenale Toxocara canis Toxocara cati Trichuris trichiura Yeasts and fungi P a t h o g e n i c yeasts a n d fungi are p r o b a b l y of less i m p o r t a n c e for the epidemiological discussions of sludge utilisation (Table XVII). They can infect humans 833 and animals, cause allergic diseases a n d / o r produce mycotoxins. But there have also been suggestions that one should not underestimate the significance of this group of micro-organisms for public health, especially in connection with the agricultural utilisation of sewage and sewage sludge. At present, the significance of these microbes in public health has not yet been properly estimated (10, 56). TABLE XVII Pathogenic yeasts and fungi to be expected in sewage and sewage sludge (10, 56) Yeasts Fungi Candida albicans Candida krusei Candida tropicalis Candida guillermondii Cryplococcus neoformans Trichosporon Aspergillus s p p . Aspergillus fumigatus Phialophora richardsii Geotrichum candidum Trichophyton s p p . Epidermophyton spp. Effects o f s e w a g e t r e a t m e n t o n p a t h o g e n s In line with our present knowledge of the effects of sewage treatment on pathogens in wastewater, it can be stated that most pathogenic agents can survive the treatment processes of wastewater, but in reduced numbers. Some of them are adsorbed to or enclosed in faecal particles and remain within sludge during the various sedimentation processes. Therefore sewage sludge is rightly described as a concentrate of pathogens (11, 15, 22, 23, 31). S U R V I V A L O F P A T H O G E N S IN T H E E N V I R O N M E N T EPIDEMIOLOGICAL SIGNIFICANCE Given that in many countries between 4 0 % and 8 0 % of sludge is disposed of in agriculture, this is a very serious aspect. The problem is all the more serious if one considers the viability of s a l m o n e l l a s in greater detail. In an extensive survey, 303 samples were examined after digested sludge was distributed on agricultural land from tankwagons in the usual way. Salmonellas were found on grass in 2 6 % of all samples taken until the fifth week. The samples were negative only after six weeks. In 5 9 % of the samples from topsoil, salmonellas persisted until the tenth week. In the crust of sludge which can be found during dry weather periods on pastures for several weeks (when the sludge is not tilled into the grass and the topsoil), 8 4 % of all samples taken until the sixteenth week contained salmonellas (55). In our own experiments, the survival of salmonellas and Ascaris ova was investigated during sludge utilisation in forestry. Salmonella senftenberg survived on and in the soil after a single application of infected sludge in summer to experimental plots of eleven different forest stands between 424 and 820 days. After application of sludge in winter the survival times were between 104 and 350 days. Ascaris ova did not survive for longer than 78 to 107 days (61). 834 Experience in Switzerland has shown that such investigations are important. In that country the farmers favour the use of sewage sludge on their pastures, and in many cases they store the sludge from sewage treatment plants together with liquid manure from their pigs or cattle in tanks on the farm before spreading it on pastures. An epidemiological causal relationship was found between the agricultural utilisation of municipal sewage sludge and salmonella infections in cattle herds. In an analysis of nearly 27,000 cases within ten years (Fig. 1), there was unequivocal accumulation of infections with salmonellas during the green fodder period. The first increase of infections was traced to the spreading of sludge during the winter and the culmination of cases in August/September to the massive spreading of sludge after hay making and the subsequent short mowing intervals. This investigation shows that it is a perversion of hygienic principles for more and more sewage to be collected and treated with more and more sophisticated methods in plants if the sludge - which consists mainly of human faeces — is then distributed over large areas without being disinfected (11). FIG. 1 S e a s o n a l distribution o f s a l m o n e l l a i s o l a t i o n s f r o m dairy cattle approx. 27,000 samples (11) The parasitic eggs in sludge create problems in areas with pasture farming because they survive on soil and plants for many months. It is known that Ascaris eggs survived for two years in soil which had been irrigated with sewage. They were even found on plants which had been irrigated with chlorinated sewage. Our knowledge of the behaviour of viruses on plants and in soil is less adequate than in the case of bacteria. But it appears that the viability of viruses in soil is of the same order as in sewage, where they survive for over 100 days. The few available investigations were made with sewage. In 2 of 25 samples, viable enterovirus was found on plants irrigated with sewage. In addition, poliovirus I survived on sewageirrigated vegetables for 36 days. The same virus survived after artificial infection on 835 plants of cabbage, pepper and tomatoes for 4, 12 and 18 days respectively. Two other groups of researchers have shown that viruses may penetrate into the root system of plants and then into the stem. They concluded that internal as well as external contamination of plants is possible (22, 23, 25, 4 1 , 63). Of all the pathogens which survive the wastewater purification processes, parasite ova have the longest survival times in the environment. Eggs of Ascaris suum survive up to fourteen years in soil, others between several months and six years. They also tend to be resistant to chemicals, e.g. 2 0 % formalin for two years, but are killed by temperatures above 55°C in approximately ten minutes. Sewage sludge and faecal excretions are the only way by which parasites specific for h u m a n beings can reach the environment (29, 56, 65). In the case of h u m a n infections with Taenia saginata the circumstances are similar to those of salmonellosis. The agricultural utilisation of sewage sludge and sewage is only one link in the epidemiological chain. But everything possible should still be done to break such a link and to work on further links like the sanitation of campgrounds, recreation areas, parking lots and picnic areas on motorways. In conclusion, it can be stated that pathogens occur in sewage and that they are enriched in sewage sludge, which raises concern not only for public health but also for domestic animals and livestock. This has been shown in Switzerland where fertilisation of pastures with sewage sludge which has not been disinfected resulted in a spread of salmonellosis in dairy cows. These animals are therefore a link in the infection cycle of h u m a n and animal salmonelloses. The economic losses caused by salmonellosis in the western states of the Federal Republic of Germany in 1977 were calculated for the sector of human medicine as 107 million DM (approx. US$ 59 million) and for livestock (cattle, calves, pigs and poultry) another 132 million DM (approx. US$ 73 million) (38). For damage caused by viruses and parasites in sewage sludge no figures are available. Connections have been proved only for Taenia saginata. In any case, the agricultural utilisation of hygienically dubious sewage sludge poses a risk for the whole national economy. This can be prevented only by sanitation measures like disinfection of sludge prior to its utilisation in agriculture. MEASURES FOR SANITATION OF SEWAGE S L U D G E To prevent damage to agriculture and public health by the utilisation of hygienically dubious sewage sludge, the C E C issued a "Directive on the use of sewage sludge in agriculture" on 12 J u n e 1986. Within three years Member States had to apply the laws, regulations and administrative provisions contained in this Directive (16). In the Federal Republic of Germany the government issued an " O r d i n a n c e on sewage s l u d g e " which went into effect on 1 April 1983. This ordinance stipulates that the use of sewage sludge on pasture and forage land is no longer allowed if it is not "hygienically s a f e " (sanitised, disinfected) (4). Since the legislators did not define the term "hygienically s a f e " , a working group was formed to elaborate a definition and also to define the appropriate technologies for achieving "hygienically safe" sludge. These definitions are presented below as an example, because they are more precise and more detailed than in other countries. 836 Definition To be sanitised (hygienically safe) according to Paragraph 2, Article 2 of the German " O r d i n a n c e on sewage sludge", a sludge has lo be treated by a sanitation process for which appropriate investigation has proved that: a) the number of indigenous or seeded salmonellas is reduced by at leasl four powers of ten (logs); and h) indigenous or seeded eggs of Ascaris are rendered non-infectious; furthermore, the sanitation process must result in a sewage sludge in one gram of which, directly after the treatment, c) no salmonellas can be detected; and ilj not more than 1,000 Enterobacteriaceae can be detected. In addition to these forms of System Control (a, b) and Process Control (c, d), a continuous Operational Control is provided for. Supervision is required so that the process conditions, which are necessary for the effective sanitation of the sewage sludge, are continuously observed. The Operational Control is performed In controllable continuous recording of the process conditions which are representative and specific for each sanitation system. Sanitation technologies According to the above definitions, six established technologies have been evaluated from the aspect of ensured sanitation. /. Sludge pasteurisation - Process (pre-pasteurisation) description During pasteurisation raw sewage sludge is to be heated to temperatures below 100°C, but at least 65°C, for at least 30 minutes. This is done prior to a stabilisation process and is known as pre-pasteurisation. Comminution of larger particles before the pasteurisation process is also necessary. To ensure that all sludge particles arc exposed to the reaction temperature and time, their size may not exceed 5 mm. Other temperature/time combinations can also be used, for example: 70°C for 25 min, 75°C for 20 min or 80°C for 10 min. Even at higher temperatures a reaction time of less than ten minutes is not allowed. — Process control The process conditions of the technology, especially: a) the temperature in the reactor; and b) the reaction time are to be recorded by continuous plotting. 837 2. Aerobic-thermophilic stabilisation of sludge (ATS) - Process description In the course of the ATS process caused by air (oxygen) supply, exothermal microbial degradation and metabolic processes result in a rise of temperature and of pH up to values of about 8. Provided that the reaction vessel is well insulated, that the air supply is correctly calculated and that the sludge has a sufficient concentration of organic dry matter, temperatures can be reached which provide for stabilisation and also sanitation of the sludge. ATS processes should be operated in two-stage reactors (two vessels connected in series) at least, to avoid the microbiological disadvantages of hydraulic shortcircuits. Detention times of at least, five days are required when both reactors have the same volume. Based on the batch-type operation (e.g. one hour feeding per day) and 23 hours stabilisation (reaction time) and of the temporary decrease of temperature inevitably connected with this type of operation, the following reaction times and temperatures are necessary: 23 h at 50 C. 10 h at 55°C or 4 h at 60"C. Process control To supervise the process conditions, the following parameters are to be recorded by continuous plotting: a) the temperatures and their reaction times in the reactors in at least two positions with recording instruments b) the pH values of the raw sludge and of the effluent of the ATS process c) the daily sludge volume flow, from which the reaction time can be calculated using known volumes of the reactors. 3. Aerobic-thermophilic stabilisation of sludge (A TS) with subsequent anaerobic digestion - Process description In the aerobic-thermophilic first stage the sludge is sanitised. The sanitation is ensured by sufficiently high temperatures in the sludge, which can be produced by additional heating with extraneous energy and exothermic microbial processes during the partial stabilisation. In the course of subsequent anaerobic, mesophilic or thermophilic stabilisation, the necessary security of the sanitation process is ensured. By treatment in this two-stage process the sludge is considered as sanitised if in the first stage either the conditions of pre-pasteurisation are fulfilled or if the reaction temperature of at least 60°C is kept continuously for at least four hours. During these four hours no raw sludge may be added. In the second (anaerobic) stage a process temperature of at least 30°C must be maintained. - Process control To supervise the process conditions, the following parameters are to be recorded by continuous plotting: a) in bj in the first stage: the temperature in two positions the reaction time the second stage: the temperature. 838 4. Treatment of sludge with lime as Ca(OH)2 (lime hydrate, — Process slaked lime) description C a ( 0 H ) 2 (calcium hydroxide, lime hydrate, slaked lime) is used for sanitation of liquid sludge before use, or for conditioning sludge before dewatering. In both cases the addition of lime increases the p H as a function of the a m o u n t of lime added and the properties of the sludge. Wet addition of lime as a suspension should be preferred to lime powder because of the better mixing and sanitising effect. The initial p H of the lime-sludge mixture must be at least 12.6 and the mixture must be stored for at least three months (reaction time) before use. — Process control T o supervise the process conditions of each batch produced, the following parameters are to be recorded: a) the initial p H value h) the reaction time. 5. Treatment of sludge with lime as CaO (quicklime, — Process unslaked lime) description By adding CaO to dewatered sludge, the lime-sludge mixture is heated to temperatures between 55°C and 70°C by exothermic reaction of the calcium oxide with available water, when the insulation is adequate. The initial pH of the limesludge mixture must reach at least 12.6 and the temperature of the whole mixture at least 55°C for two h o u r s . — Process control T o supervise the process conditions of each batch produced, the following parameters are to be recorded: a) the mixture ratio of lime and dry matter of sludge b) the initial p H of the lime-sludge mixture c) the temperature at least two hours after mixing in three positions, one of which must be in the outer zone of the mixture. 6. Composting — Process of sludge in windrows description The sanitation of sludge by composting in windrows with bulking material (e.g. municipal refuse, straw, sawdust, wood shavings) is caused by the heat generated during composting by microbial processes. Besides this temperature and the reaction time, microbial metabolic substances having antibiotic effects are also of importance. For the sanitising effect to occur, it is necessary to have a sufficient aeration of the mixture of sludge and bulking material by technical means, for example, by turning the heaps or by forced aeration (for windrows or piles which are not turned). The effective temperatures must prevail in each part of the composting material for the necessary reaction time. 839 The initial water content of the composting material must be 40-60% and the reaction temperature in the heap at least 55°C for three weeks. - Process control To supervise the process conditions for each windrow, the following parameters are to be recorded: a) the initial water content of the mixture b) the temperature measured at least daily in three positions and at different distances from the surface of the heap; one position in the central part and one in the outer zone c) the composting time and turning (number, date). 7. Composting - Process of sludge in reactors description The sanitising of sludge by composting in reactors (in-vessel composting) with bulking material (e.g. sawdust, wood shavings, bark, reflux material) is caused by the heat generated during composting by microbial processes. Besides temperature and reaction time, microbial metabolic substances having antibiotic effects are also of importance. For the sanitising effect to occur, it is necessary to have sufficient aeration the mixture of sludge and bulking material by technical means. The steadiness desired temperature profiles in the reactors can be influenced and controlled techniques of aeration, filling and emptying. Effective temperatures must prevail each part of the composting material for the necessary reaction time. of of by in The initial water content of the composting material should not exceed 7 0 % . The complete mixture should be exposed to a temperature of at least 55 °C during a passage time through the reactor of at least ten days. In addition, the composting material should not pass the " h o t z o n e " with a temperature of at least 65°C sooner than in 48 h o u r s . T h e reactor passage shall be followed by a phase of curing the material in heaps or piles for at least two weeks with at least one turning, or in a second reactor to provide the necessary security for the sanitation process. Successful sanitation depends to a large extent on an undisturbed operation of the composting process. W h e n disturbances of operations are connected with a decrease in temperature, the batch of compost must either be used as reflux material and thus pass the reactor a second time, or must be composted again in a heap under the conditions described for composting of sludge in heaps. - Process control T o supervise the process conditions, the following parameters are to be recorded: a) the initial water content of the mixture b) the temperature in at least three positions: one before the compost reaches the " h o t z o n e " , one in the " h o t z o n e " and at least one in the peripheral zone of the reactor 840 c) the storage time and turning of the curing heaps (date, number) or reaction time in the second reactor d) disturbances of operations (causes, duration). To make it easier to assess the sanitising effect of thermal technologies, the time/temperature combinations are summarised in Fig. 2. These ensure the destruction of some selected pathogens. The lines in the diagram represent the carefully evaluated upper limits for the thermal destruction of the mentioned pathogen, which requires estimation of the particular time/temperature combinations. A sanitation technology whose time/temperature effects lie in the "safety z o n e " should destroy all pathogens in the treated sludge. The combinations shown in Fig. 2 which mark the border of the "safety z o n e " are at least: one hour at > 6 2 ° C , one day at > 5 0 ° C or one week at > 4 6 ° C . For further details concerning other pathogens see the original publication (25). FIG. 2 Effect o f t e m p e r a t u r e a n d time o n s o m e selected p a t h o g e n s isolated f r o m s e w a g e s l u d g e and septic t a n k s (25) 841 SURVIE DES MICRO-ORGANISMES P A T H O G È N E S ET DES PARASITES D A N S LES DÉJECTIONS A N I M A L E S , LE FUMIER ET LES BOUES D ' É P U R A T I O N . - D . Strauch. Résumé: De nombreux agents pathogènes responsables de maladies infectieuses sont présents dans les sécrétions ou les déjections animales, fécales ou autres. Certains agents sont également excrétés par des animaux cliniquement sains, ou victimes d'infections latentes, dans le cas de maladies contagieuses à étiologie multiple. Dans les différents élevages, les germes pathogènes gagnent les installations qui collectent les excréments en vue de leur utilisation comme fertilisants, sous forme de fumier ou de lisier. Dans ces conditions, les éleveurs ne sont pas conscients de la présence des germes pathogènes dans le fumier, et ne prennent donc pas les mesures nécessaires à la prévention des maladies qu'entraîne son utilisation. Les germes pathogènes ne survivent que difficilement dans le fumier de ferme, du fait des réactions thermiques, biologiques et biochimiques qui se produisent au sein de ces matières organiques. Par contre, dans le cas du lisier la température reste stable et l'activité biochimique réduite, ce qui favorise la survie des germes pathogènes. Il est donc nécessaire de prendre certaines précautions lors de l'utilisation du fumier et du lisier comme engrais. L'utilisation par les agriculteurs des boues provenant des eaux usées des agglomérations urbaines est courante dans de nombreux pays. Ces boues véhiculent cependant des germes pathogènes d'origine humaine, qui ne seront pas totalement éliminés par les procédés d'épuration. Au contraire, la sédimentation des dépôts favorisera leur multiplication. Afin de protéger le bétail, Il est donc nécessaire de traiter les boues potentiellement contaminées avant de les utiliser comme engrais dans les fermes. L'auteur décrit les conséquences épidémiologiques de l'utilisation des boues d'épuration à des fins agricoles, ainsi que tes traitements possibles de ces boues. MOTS-CLÉS : Bactéries - Bétail - Boues d'épuration - Désinfection Epidémiologie - Fumier - Lisier - Parasites - Résistance - Virus. SUPERVIVENCIA D E MICROORGANISMOS P A T Ó G E N O S Y P A R Á S I T O S EN DEYECCIONES A N I M A L E S , ESTIÉRCOL Y LODOS D E A L B A Ñ A L . - D . Strauch. Resumen: Los agentes causantes de numerosas enfermedades infecciosas son excretados por vía fecal y otras secreciones del organismo. Algunos agentes patógenos también son excretados por animales clínicamente sanos o que sufren de infecciones latentes, en caso de enfermedades transmisibles de etiología múltiple. En todos los establecimientos pecuarios, los patógenos llegan al suelo a través de ¡as instalaciones que recogen el estiércol en forma sólida o líquida. En tales condiciones, los productores no se clan cuenta de que el estiércol puede contener agentes patógenos y no toman las precauciones necesarias para evitar la difusión de enfermedades a través de su uso. Los patógenos no sobreviven mucho tiempo en el estiércol almacenado en las granjas, debido a las reacciones térmicas, biológicas y bioquímicas que se producen en estas materias orgánicas. Sin embargo, en el caso del estiércol licuado, la temperatura es estable y la actividad bioquímica reducida, lo que favorece la supervivencia de los gérmenes patógenos. Por lo tanto, para evitar la difusión de enfermedades por el uso de estiércol como fertilizante, es necesario tomar ciertas precauciones. El uso agrícola de ios lodos de albañal urbano es corriente en muchos países, pero dichos lodos contienen agentes patógenos de origen humano que no llegan 842 a ser destruidos totalmente por los procedimientos de depuración, sino que, por el contrario, son multiplicados por la sedimentación de los depósitos. Por consiguiente, para proteger al ganado, es necesario tratar los lodos que puedan estar contaminados antes de utilizarlos como fertilizantes o abonos en las granjas. 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